Praeparatum, Testing And Quality Analysis Of N-V Proteinase

Thrombotic disease is a kind of frequent disease to threaten mankind health in modern time. It has considerable practical importance for treating this disease to develop neotype thrombolytic drug. The thrombolytic therapy and thrombus enzyme is the focus of the study treatment now. The commonly used thrombolytic therapy in clinic has developped three generation. The first generation has disadvantages:for example ,to lack of fibrinolysis specificity,to have strong hemorrhage side effect, to hard to control dosage. The second generation has some fibrinolysis specificity,but, has strong hemorrhage side effect and short half life.The third generation is mainly improved product by protein engineering medium and molecular biology,but occupying experiment stage at present. on the whole,all these thrombolytics exist some defects, needing to develop a new type safe medicine.N-V protease is a new thrombolytic drug from Marine organisms Nereis, developed by Jilin University Professor of Biochemistry Department of Hong Group, Used technology related chromatography.It is found that this proteinum has Fiber protease, activity, but Plasminogen activator,s role.It can hydrolysis fibrin Directly, Plays a role early,do not accumulate in the body, has Fewer side effects, demonstrats good prospects In the treatment of thrombotic diseases. It Has formed a package of Purification Process at present, the purity of the extracted product meets the requirements of the Ministry of Health declared drug standards. The registration of the drug need accurate calibration for purity, composition and structure.In order to grope the optimum chromatographic condition, wo analysis N-V protease finished product with two distinct principle high-performance liquid chromatography (HPLC),which verify and supply for each other. First, wo use gel pattern chromatographic column(tsk-gel G2000SW <7.5mm×30.0cm>)to find out three main influential factors : composition,pH and ionic strength of the moving phase ,from several influential factors .We hold other chromatographic condition and change the three main influential factors of the moving phase .And then analyze the Samples ,using the optimized condition combined the fibronolysis activity detection together. Second, wo use ion exchange chromatographic column(DEAE-5PW<7.5mm×7.5cm>), beside the above three main influential factors, still considering elution program and other conditions. Establishing the chromatographic condition,wo collect the sample of every hump by each,and detect the activity ,do double immunodiffusion,do PAGE electrophoresis and make SDS-PAGE electro- phoretic analysis. Collect the different sample of every hump from DEAE- 5PW column,retransfuse gel column and analysis it.This experiment aims at groping optimum condition on analyzing N-V protease by HPLC,and preparing condition for reporting to a higher authority. The suitable chromatographic separation condition for gel chromato- graphy: flow rate 1ml/min,column temperature 20℃,sample quantity 10μg,detection wave length 214nm and 280nm,mobile phase constitting of NaCl and NH4HCO4 ,concentration 0.1 mol/L,0.05mol/L。 For ion exchange chromatographic column , the mobile phase is four different buffer solutions. Na2HPO4-citrate buffer solution,PB buffer solution, Tris-Hcl buffer solution, acetate buffer solution.the better candidate is natrium aceticum-acetic acid buffer(pH=6.7),0～0.3mol/L Nacl linear gradient elution, flow rate 1.0ml/min, column temperature 20℃,sample size 20μg,detection wave length 214nm and 280nm.The suitable chromatographic condition has been established in his experiment. Using gel chromatography ,these are three peaks, the retention times are 7.34 min,8.46 min,9.96 min。Using ion exchange chromatography, these are five peaks,the retention times are 2.76min,7.35min,14.16min,16.94min,21.19min.All the peaks have better resolving power.After handling with N-V protease, detecting and comparing anteropos- terior spectrogram, we find that the enzyme is a polymerization proteinum that has identical subunits,this result is different from discovered organism thrombolytics.Three peaks can be obtained by gel filtration, five peaks can be obtained by chromatography, they all have Biological activity,and Immunoprecipitate by immunodiffusion.The five peaks that collected by HPIEC is analyzed by SEC-HPLC then ,the result is satisfactory .The working sample has reproducibility ,the degree of accuracy and recovery rate is in line with related regulation.When the sample is freeze-dried, the peak shape of the ter-peak appears evident variance between the freeze-dried sample including excipient and that unincluding excipient. So direct gelsiccation can destroy the sample protein.By testing dextran and mannitol , the suitable excipient is dextran,and the suitable addition is 10mg per 20mg product.